Pajares, M., Jimenez Moreno, N., García-Yagüe, A. J., Escoll, M., de
Ceballos, M. L., Van Leuven, F., Ràbano, A., Yamamoto, M., Rojo, A.
I., & Cuadrado, A. (2016). Transcription factor NFE2L2/NRF2 is a
regulator of macroautophagy genes.
Autophagy
,
12
(10), 1902-1916.
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Transcription factor NFE2L2/NRF2 is a regulator of
macroautophagy genes
Marta Pajares, Natalia Jiménez-Moreno, Ángel J. García-Yagüe, Maribel
Escoll, María L. de Ceballos, Fred Van Leuven, Alberto Rábano, Masayuki
Yamamoto, Ana I. Rojo & Antonio Cuadrado
To cite this article: Marta Pajares, Natalia Jiménez-Moreno, Ángel J. García-Yagüe, Maribel
Escoll, María L. de Ceballos, Fred Van Leuven, Alberto Rábano, Masayuki Yamamoto, Ana
I. Rojo & Antonio Cuadrado (2016) Transcription factor NFE2L2/NRF2 is a regulator of
macroautophagy genes, Autophagy, 12:10, 1902-1916, DOI: 10.1080/15548627.2016.1208889
To link to this article: http://dx.doi.org/10.1080/15548627.2016.1208889
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BASIC RESEARCH PAPER
Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes
Marta Pajares
a
,
b
, Natalia Jim
enez-Moreno
a
,
b
,
c
,
Angel J. Garc
ıa-Yag
€
ue
a
,
b
, Maribel Escoll
a
,
b
, Mar
ıa L. de Ceballos
b
,
d
,
Fred Van Leuven
e
, Alberto R
abano
f
, Masayuki Yamamoto
g
, Ana I. Rojo
a
,
b
, and Antonio Cuadrado
a
,
b
a
Instituto de Investigaciones Biom
edicas “Alberto Sols” UAM-CSIC, Instituto de Investigaci
on Sanitaria La Paz (IdiPaz) and Department of Biochemistry,
Faculty of Medicine, Autonomous University of Madrid, Madrid, Spain;
b
Centro de Investigaci
on Biom
edica en Red sobre Enfermedades
Neurodegenerativas (CIBERNED), ISCIII, Madrid, Spain;
c
Present address: School of Biochemistry, University of Bristol, Medical Sciences Building,
University Walk, Bristol, UK;
d
Neurodegeneration Group, Department of Cellular, Molecular and Developmental Neurobiology, Instituto Cajal, Consejo
Superior de Investigaciones Cient
ıficas, Madrid, Spain;
e
Experimental Genetics Group-LEGTEGG, Department of Human Genetics, KU Leuven, Leuven,
Belgium;
f
Department of Neuropathology and Tissue Bank, Unidad de Investigaci
on Proyecto Alzheimer, Fundaci
on CIEN, Instituto de Salud Carlos III,
Madrid, Spain;
g
Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan
ARTICLE HISTORY
Received 10 August 2015
Revised 16 June 2016
Accepted 27 June 2016
ABSTRACT
Autophagy is a highly coordinated process that is controlled at several levels including transcriptional
regulation. Here, we identify the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) as a
regulator of autophagy gene expression and its relevance in a mouse model of Alzheimer disease (AD)
that reproduces impaired APP (amyloid b precursor protein) and human (Hs)MAPT/TAU processing,
clearance and aggregation. We screened the chromatin immunoprecipitation database ENCODE for 2
proteins, MAFK and BACH1, that bind the NFE2L2-regulated enhancer antioxidant response element (ARE).
Using a script generated from the JASPAR’s consensus ARE sequence, we identified 27 putative AREs in 16
autophagy-related genes. Twelve of these sequences were validated as NFE2L2 regulated AREs in 9
autophagy genes by additional ChIP assays and quantitative RT-PCR on human and mouse cells after
NFE2L2 activation with sulforaphane. Mouse embryo fibroblasts of nfe2l2-knockout mice exhibited
reduced expression of autophagy genes, which was rescued by an NFE2L2 expressing lentivirus, and
impaired autophagy flux when exposed to hydrogen peroxide. NFE2L2-deficient mice co-expressing
HsAPP
V717I
and HsMAPT
P301L
, exhibited more intracellular aggregates of these proteins and reduced
neuronal levels of SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5 and GABARAPL1. Also, colocalization of
HsAPP
V717I
and HsMAPT
P301L
with the NFE2L2-regulated autophagy marker SQSTM1/p62 was reduced in
the absence of NFE2L2. In AD patients, neurons expressing high levels of APP or MAPT also expressed
SQSTM1/p62 and nuclear NFE2L2, suggesting their attempt to degrade intraneuronal aggregates through
autophagy. This study shows that NFE2L2 modulates autophagy gene expression and suggests a new
strategy to combat proteinopathies.
KEYWORDS
Alzheimer disease; amyloid
precursor protein;
neurodegenerative diseases;
neuroprotection; oxidative
stress; proteostasis; Tau
Introduction
The homeostatic control of protein synthesis, folding, trafficking
and degradation, i.e. proteostasis, is critical for biological pro-
cesses, and its alterations lead to pathological outcome, including
neurodegenerative diseases.
1
The 2 main mechanisms that con-
trol protein degradation are the 20S and 26S proteasome and
autophagy in its 3 primary modalities, microautophagy, chaper-
one-mediated autophagy and macroautophagy.
2
These processes
ensure timely degradation of misfolded, oxidized or altered pro-
teins that otherwise develop proteinopathy. In particular, macro-
autophagy, usually termed autophagy, participates in the
degradation of long-lived proteins that, if improperly cleared,
aggregate and cause cellular damage.
The autophagy process requires the coordinated participation
of a set of structural proteins that participate in the formation of
autophagosomes and autolysosomes, as well as cargo-selective
proteins that recognize specific cargos and direct them to degra-
dation.
3
Most studies on the regulation of macroautophagy have
focused on protein-protein interactions based on MTOR-depen-
dent mechanisms. For instance, upon serum-deprivation or inhi-
bition by rapamycin, low MTOR activity allows ULK1 activation
by disruption of the interaction with AMP kinase and results in
increased autophagy.
4
Because these regulatory mechanisms are
cell signaling dependent, they probably represent a quick adapta-
tion to cellular needs of a specific moment. Other reports indi-
cate positive
5-8
and negative
9,10
transcriptional regulation of
autophagy, which might represent a long-term mechanism of
adaptation. Two documented transcription factors that fulfill this
role are the helix-loop-helix transcription factor TFEB and the
forkhead transcription factor FOXO.
11,12
Given the complexity
CONTACT Ana I. Rojo airojo@iib.uam.es Instituto de Investigaciones Biom
edicas “Alberto Sols”, UAM-CSIC C/ Arturo Duperier 4 28029 Madrid, Spain;
Antonio Cuadrado
antonio.cuadrado@uam.es Instituto de Investigaciones Biom
edicas “Alberto Sols”, UAM-CSIC C/ Arturo Duperier 4 28029 Madrid, Spain.
Color versions of one or more of the figures in the article can be found online at
www.tandfonline.com/kaup.
Supplemental data for this article can be accessed on the publisher’s website.
© 2016 Marta Pajares, Natalia Jim
enez-Moreno,
Angel J. Garc
ıa-Yag
€
ue, Maribel Escoll, Mar
ıa L. de Ceballos, Fred Van Leuven, Alberto R
abano, Masayuki Yamamoto, Ana I. Rojo, and Antonio
Cuadrado. Published with license by Taylor & Francis.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (
http://creativecommons.org/licenses/by-nc/3.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
AUTOPHAGY
2016, VOL. 12, NO. 10, 1902–1916
http://dx.doi.org/10.1080/15548627.2016.1208889
of the macroautophagy system and the need for a coordinated
interplay between autophagy-regulated and -regulating proteins,
it is hypothesized that the expression of autophagy genes is con-
nected through additional common regulatory transcription fac-
tor(s).
Considering that autophagy is a proteostatic and defensive
mechanism, we sought to determine if this process is regulated
by NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2). The
transcription factor NFE2L2 is considered a master regulator
of cellular homeostasis, as it controls the expression of numer-
ous cytoprotective genes.
13,14
These genes share a common cis-
acting element in their promoters, named the antioxidant
response element (ARE), and the list of cytoprotective genes
that contain this enhancer is continuously growing to include
those involved in biotransformation, antioxidant defense, and
metabolic reprogramming. Regarding autophagy, the cargo
recognition protein SQSTM1/p62 contains the motif KIR that
interacts with KEAP1 leading to its transport into the phago-
phore (the autophagosome precursor) and relieving NFE2L2
from inhibition.
15,16
In connection with this finding, SQSTM1
contains one ARE and is therefore upregulated by NFE2L2,
creating a feed-forward cycle.
17
The cargo recognition protein
CALCOCO2/NDP52 also appears to be regulated by NFE2L2,
leading to the clearance of toxic proteins such as phosphory-
lated HsMAPT (p-MAPT) in a mouse model of AD, while 3
different ARE sequences were identified in its promoter
region.
18
Impaired proteostasis is a crucial hallm ark of neurodegener-
ative diseases.
19-21
Protein pathology in AD is related to the
altered processing of APP, which leads to intracellular accu mu-
lation and extracellular deposition of amyloid-b (Ab) peptides,
as well as agg regation of p-MAPT into intracellular neurofibril-
lary tangles.
22
Both APP and HsMAPT are degraded, at least in
part, through autophagy and several reports suggest that this
process is altered in AD. For instance, dystrop hic neuri tes of
AD patients contain more autop hagosomes,
23
increased levels
of MTOR signaling components and lysosomal hydrolases.
24
In this study, we sought to determine if genes that partici-
pate in autophagy are regulated by NFE2L2. Our results sup-
port our hypothesis that NFE2L2 participates in the regulation
of autophagy gene expression and suggest a new therapeutic
strategy for proteinopathies based on the activation of this tran-
scription factor.
Results
Identification of putative AREs in autophagy genes
To define comprehensively the role of NFE2L 2 in the transcrip-
tional regulation of autophagy, we searched the Encyclopedia of
DNA Elements at UCSC (ENCO DE)
25
of the human genome
(Feb. 2009) for autophagy genes with putative AREs. This data-
base contains the experimental data from chromatin immuno-
precipitation (ChIP) studies of several transcription factors.
Although NFE2L2 is not included, we analyzed 2 other ARE
binding factors, MAFK and BACH1, for which information is
available. We found evidence of MAFK or BACH1 binding in
many genes involved directly or indirectly in autophagy. Then,
we developed a Python-based bioinformatics analysis script, to
compare the consensus human ARE from the JASPAR data-
base
26
with putative AREs in the promoter regions of these
genes (for details see Material and methods and Tables S1 and
S2). We detected 25 putative new AREs (relative score over
80%) in these 16 autophagy genes, besides the internal controls
that our algorithm retrieved as the bona fide ARE-genes in
NQO1 and HMOX1 (
Table 1). Some of these genes, such as
SQSTM1
17
or CALCOCO2
18
were reported to be regulated by
NFE2L2, and for SQSTM1 we identified the previously
described ARE
17
plus 3 new sequences. The other autophagy
ARE-genes were identified here for the first time. Moreover,
several putative AREs are conserved in mice (Tables S1–S3).
Validation of putative AREs by ChIP and qRT-PCR
The newly identified ARE sequences were subsequently vali-
dated by ChIP assays for NFE2L2. Because of the lack of ade-
quate antibodies to immunoprecipitate endogenous NFE2L2
efficiently, we used HEK293T cells transfected with an expres-
sion vector for V5-tagged NFE2L2. Moreover, this construct
lacked the KEAP1 regulatory domain (ETGE), facilitating
NFE2L2 stabilization, its translocation to the nucleus and
binding to target genes.
27
ChIPs were performed with anti-V5
antibody, and with anti-IgG as negative control. Immunopre-
cipitated DNA was analyzed by quantitative real time PCR
(qRT-PCR) with specific primers surrounding the putative
AREs (Table S4).
Table 1. Putative Antioxidant Response Elements (AREs) in the promoter regions
of autophagy genes with a relative score higher than 80%. The table also shows
the max score and the localization in the human genome.
GENE
(human)
Localization in
the human genome
Max
score
Relative
score
ARE putative
binding sequence
SQSTM1 chr5:179247562-179247551 19.11 1.0 ATGACTCAGCA (1)
chr5:179246594-179246583 18.97 0.997 GTGACTCAGCA
(2)
chr5:179247479-179247490 11.44 0.816 GCGACCTAGCA (3)
chr5:179246116-179246105 13.39 0.863 GTGAGTCAGCG (4)
CALCOCO2 chr17:46906385-46906374 12.65 0.845 ATGAGTAAGCC
chr17:46906430-46906441 12.34 0.838 GTGAGGAAGCA
ULK1 chr12:132381043-132381032 12.62 0.844 ATTAGTCAGCA
ULK2 chr17:19783789-19783800 15.74 0.919 GTGACAGAGCA
ATG2B chr14:96849918-96849929 11.38 0.815 CTGCCTAAGCA
ATG4C chr1:63212369-63212380 13.03 0.854 ATGAAATAGCA
ATG4D chr19:10652326-10652315 14.96 0.9 ATGACAATGCA
chr19:10655902-10655891 10.94 0.804 GTGACTTTGAA
ATG5 chr6:106873861-106873872 11.01 0.806 CTGACCTTGCA
ATG7 chr3:11347170-11347159 15.94 0.924 ATGAGTCAGCA
chr3:11320731-11320720 13.65 0.869 CTGACTCAGCT
chr3:11347216-11347227 11.08 0.807 AGGACTTGGCA
chr3:11320599-11320610 11.07 0.807 ATGATTGAGCT
GABARAPL1 chr12:10364974-10364963 14.58 0.891 GTGACTCAGGA
chr12:10366072-10366083 18.97 0.997 GTGACTCAGCA
ATG9B chr7:150723213-150723224 14.24 0.883 CTGACTCAGCC
ATG10 chr5:81278192-81278181 16.65 0.941 CTGACTCAGCA
ATG16L1 chr2:234134609-234134598 16.53 0.938 ATGATTCAGCA
chr2:234134578-234134589 13.56 0.867 ATGACCTAGCC
WIPI2 chr7:5239766-5239755 16.65 0.941 ATGACTGAGCA
LAMP1 chr13:113951364-113951353 12.06 0.831 GTGACCCGGCC
AMBRA1 chr11:46576251-46576240 11.51 0.818 TTGACTTAGCT
chr11:46572300-46572311 10.84 0.802 ATGACTAATCT
NQO1 chr16:69760919-69760908 18.97 0.997 GTGACTCAGCA
HMOX1 chr22:35768205-35768216 12.3 0.837 ATGCCTCAGCC
chr22:35768021-35768010 18.97 0.997 GTGACTCAGCA
chr22:35768127-35768116 18.97 0.997 GTGACTCAGCA
chr22:35747111-35747100 13.24 0.859 GTTACTCAGCC
Asterisks denote AREs that have been described previously.
AUTOPHAGY
1903
Among the 27 putative AREs analyzed in autophagy genes we
found enrichment of 11 ARE regions in V5-immunoprecipitated
chromatin, indicating that NFE2L2 binds these promoter regions
(
Fig. 1A-B). Our study also detected enrichment of 2 positive
controls from the bona fide NFE2L2 targets NQO1 and HMOX1.
No enrichment was detected with specificprimersforACTB,for
an upstream region of NQO1 that does not contain AREs,
28
or
for ATG3, all of which had low scores for putative AREs in the
bioinformatics analysis. Negative control assays were further per-
formed without antibodies or on nontransfected cells (data not
shown). Next, we sought to determine the existence of other
potentialAREsintheSQSTM1 promoter aside from the one
already described.
17
For this purpose we used specificprimers
surrounding the other potential AREs (Table S4 and
Table 1).
We observed that NFE2L2 bound to all these sequences
(
Fig. 1B), demonstrating that they constitute additional AREs in
this gene.
We went on to further confirm our observations in
HEK293T cells, treated with the NFE2L2 activator sulforaph-
ane (SFN) (15 mM, 12 h), which has been used previously to
induce autophagy.
29-31
Transcript levels of the selected auto-
phagy genes were analyzed by qRT-PCR, demonstrating
increased expression of SQSTM1, CALCOCO2, ULK1, ATG2B,
ATG4D, ATG5, and GABARAPL1 upon SFN treatment
(
Fig. 1C). HMOX1 was analyzed and confirmed as a positive
control. We next extended our observations to murine cells by
analyzing hippocampus-derived HT22 cells treated with SFN
(15 mM, 12 h). SFN augmented the expression of all the murine
counterparts of the genes identified in the human cells
(
Fig. 1D). The combined results indicated that the regulation of
expression of autophagy genes is conserved in both species.
We next analyzed the expression of these genes in mouse
embryonic fibroblasts (MEFs) from wild-type (Nfe2l2-WT) or
Nfe2l2-knockout (Nfe2l2-KO) mice by qRT-PCR (
Fig. 2A).
Impaired expression of Sqstm1, Calcoco2, Ulk1, Atg2b, Atg4d,
Atg5, Atg7 and Gabarapl1 was observed in Nfe2l2-KO MEFs.
Moreover, reduce d expression of Sqstm1, Calcoco2 and Atg7
products was also con firmed at the protein level with available
antibodies (
Fig. 2B-C).
To further define the role of NFE2L2 in the regulation of
these autophagy genes we performed chemical and genetic
manipulations of this transc ription factor. In response to SFN
nfe2l2-KO MEFs exhibited a reduced induction of these genes
compared to Nfe2l2-WT MEFs (
Fig. 2D). In addition, rescue
experiments in nfe2 l2-KO MEFs employing NFE2L2-
DEGTE
-V5
augmented normal basal levels of expression of autophagy
Figure 1. NFE2L2 modulates autophagy gene expression. (A) HEK293T cells were transfected with an expression vector for NFE2L2
DETGE
-V5.
27
ChIP analysis was performed
with anti-IgG or anti-V5 antibodies and the potential AREs with the highest score were analyzed by qRT-PCR. (B) The same ChIP analysis of putative AREs in the promoter
of SQSTM1. The figures show representative data normalized as the fold of enrichment with the anti-V5 antibody vs. the IgG antibody. In (A), ACTB and an upstream region
of NQO1 that does not contain any ARE (NQO1
) were analyzed as negative controls. Previously described AREs in HMOX1, NQO1, SQSTM1 and CALCOCO2 were analyzed as
positive controls. These experiments were repeated 3 times with similar results. In (B), numbers in brackets indicate the AREs from
Table 1 specifically amplified. (C and D)
HEK293T and HT22 cells were submitted to sulforaphane (SFN, 15 mM) for 12 h. mRNA levels of the indicated genes were determined by qRT-PCR and normalized by Actb
levels. Data are mean § SEM (n D 3). Statistical analysis was performed with the Student t test.
,p< 0.05;
,p< 0.01; and
,p< 0.001 vs. untreated conditions.
1904
M. PAJARES ET AL.